Purpose: To ensure the efficiency and safety of transplanted human embryonic stem cell (hESC)-derived osteoblast-like cells (hESC-OS) for bone regeneration, this study was designed to determine the effects of continuous cell expansion on the osteoblastic differentiation stability, pluripotency, and tumorigenic potential of long-term expanded hESC-OS. Methods: hESCs manually harvested as cell aggregates or enzymatically dissociated as single cells were directly incubated in osteogenic medium and serially passaged to passage 25. Expression of osteoblast-related genes, pluripotent regulator genes, and genes related to tumorigenesis were examined at the primary passage and every 5 passages thereafter. hESC-OS were subcutaneously transplanted into nude mice for 4–24 weeks to test for teratoma formation. hESC-OS were recultivated in hESC culture conditions to evaluate the extent to which reverse differentiation back to the undifferentiated stage may occur. Results: hESC-OS derived from hESC aggregates and dissociated cells exhibited comparable osteoblast differentiation patterns. Expression levels of osteoblast-related genes reached plateau levels at passages 5–10 before declining in higher passages. Expression of tumor-associated genes was not significantly increased. Only hESC-OS at primary and first passages formed teratomas after 4 weeks in vivo. The hESC-OS were not able to revert to hESCs. Conclusions: Expanded hESC-OS demonstrated lineage-specific differentiation stability, did not maintain the pluripotency of hES cells, and were genetically stable. Thus, hESC-OS may be considered for large animal preclinical studies.

Keywords:
Human embryonic stem cells, Osteoblastic differentiation, Tumorigenesis, Transdifferentiation
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© 2011 S. Karger AG, Basel
2011
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